The immediate goal of this project continues to be the establishment of high titered scrapie infected tissue culture cell lines. Scrapie is a naturally occurring spongiform encephalopathy of sheep and goats which causes clinical and pathological changes similar to those of Creutzfeldt-Jakob and Kuru diseases of man. Scrapie grows to high titer in mouse lymphoid tissue and brain but has never been passaged at high titer in any in vitro system. Adaptation of the agent to cell culture will permit detailed characterization of the agent(s) and provide a system for better definition of the pathogenesis of these diseases. We have utilized several approaches designed to provide infected in vitro cell lines. In one approach scrapie infected mice were injected with one of several splenotropic tumor cell lines. Once tumors were established in vivo, they were explanted, maintained in vitro and assayed for the presence of scrapie agent. Eight tumors representing several cell types were used. Results obtained during the past year indicated that no persistently infected cell lines were established using this protocol. A second approach utilized hybridoma technology. Spleen cells from scrapie infected mice were fused with a myeloma cell line. If a scrapie infected cell was a partner in the fusion, persistently infected lines might result. However, no positive cultures were identified. Another approach was begun utilizing a neuroblastoma cell line known to support the replication of at least one isolate of Creutzfeld-Jakob agent. A second neuroblastoma line known to support the replication of rabies virus was also utilized. We have attempted to infect these cell lines in vitro with purified scrapie material obtained from scrapie infected mice or from scrapie infected hamsters.